Project Summary Granulocyte transfusion (GTX) has been utilized as a therapeutic approach for the treatment of life-threatening bacterial and fungal infections in severely neutropenic patients. However, its clinical outcome is often hampered by inefficiency of recruitment to sites of inflammation, rapid in vivo death, and poor pathogen killing capability of transplanted neutrophils. The ultimate goal of the proposed research is to identify and characterize cellular and molecular events that can improve neutrophil performance during granulocyte transfusion. In the last funding period, we demonstrated that elevating intracellular PtdIns(3,4,5)P3 signaling pathway by disrupting PTEN enhances neutrophil function, augments bacterial clearance, and reduces mortality rate in a murine model of neutropenia-associated pneumonia. We further revealed that PTEN disruption in transfused neutrophils significantly improves the efficacy of granulocyte transfusion. These findings confirm the validity of the PtdIns(3,4,5)P3 pathway as a therapeutic target for improving the clinical outcome of granulocyte transfusion. Nevertheless, PTEN is a well-known tumor suppressor and its disruption has been implicated in tumorigenesis of numerous solid and hematologic cancers, which renders it unsuitable as a therapeutic target. Recently, we reported that PtdIns(3,4,5)P3 signal in neutrophils can also be elevated by disrupting InsP6K1, an enzyme responsible for the synthesis of InsP7, a cytosolic molecule that negatively regulates PtdIns(3,4,5)P3 signaling. InsP6K1 deficient neutrophils possess an enhanced bacteria killing capability and their recruitment to the site of inflammation is augmented. Importantly, homozygous InsP6K1 KO mice are viable and do not display any gross physical or behavioral abnormalities. No tumors of any kind is discovered in these mice. Based on these intriguing results, we hypothesize that disruption of InsP6K1 should be a safer, yet equally effective therapeutic strategy for improving granulocyte transfusion. In this proposed study, we will use a mouse neutropenia-related pneumonia model to test this hypothesis. First, we will investigate whether disrupting InsP6K1 can elevate PtdIns(3,4,5)P3 signal and enhance the accumulation (including recruitment and survival) of transfused neutrophils in neutropenia-related E.coli pneumonia (Aim I). In addition, we will determine whether disrupting InsP6K1 in transfused neutrophils can ultimately enhance the host defense in (Aim II), and alleviate the severity of (Aim III) neutropenia-related bacterial and fungal pneumonia. Finally, for future clinical intervention, inhibition of InsP6K1 will most likely be achieved by using chemical compounds. Thus we will examine whether pre-treatment with an InsP6K inhibitor, TNP, can also augment the efficacy of granulocyte transfusion in neutropenia-related pneumonia (Aim IV). Thus, in accordance with the general theme of the PPG, this study will provide novel therapeutic targets and/or strategies for improving the performance of neutrophils in granulocyte transfusion and therefore alleviating the severity of neutropenia-related infection in the peri-transplant setting.